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Abstract

Background

Female sperm storage has evolved independently multiple times among vertebrates to
control reproduction in response to the environment. In internally fertilising amphibians,
female salamanders store sperm in cloacal spermathecae, whereas among anurans sperm
storage in oviducts is known only in tailed frogs. Facilitated through extensive field
sampling following historical observations we tested for sperm storing structures
in the female urogenital tract of fossorial, tropical caecilian amphibians.

Findings

In the oviparous Ichthyophis cf. kohtaoensis, aggregated sperm were present in a distinct region of the posterior oviduct but
not in the cloaca in six out of seven vitellogenic females prior to oviposition. Spermatozoa
were found most abundantly between the mucosal folds. In relation to the reproductive
status decreased amounts of sperm were present in gravid females compared to pre-ovulatory
females. Sperm were absent in females past oviposition.

Conclusions

Our findings indicate short-term oviductal sperm storage in the oviparous Ichthyophis cf. kohtaoensis. We assume that in female caecilians exhibiting high levels of parental investment
sperm storage has evolved in order to optimally coordinate reproductive events and
to increase fitness.

Keywords:

Reproduction; Sperm storage; Amphibians; Caecilians

Background

Animal reproductive strategies include variable modes of sperm transfer, fertilization,
and type of offspring development. In particular female sperm storage, where male
spermatozoa remain in the reproductive tract after mating until used for fertilization,
has evolved independently and repeatedly in metazoans as a mechanism to temporarily
decouple insemination from fertilization [1,2]. In vertebrates, female sperm storage in dedicated structures occurs in all major
lineages with durations ranging from a few hours or days in most mammals (not including
bats) to long-term storage up to months in sharks, turtles, birds and also reptiles
with a reported maximum of seven years [2]. Among modern amphibians many female salamanders can store sperm in unique cloacal
spermathecae [3] and internal fertilising anurans such as tailed frogs (Ascaphus ssp.) have sperm storage in the oviducts [4]. This raises the question whether female sperm storage has also evolved in the third
group of extant amphibians, the limbless caecilians [5,6].

Caecilians perform internal fertilization with the aid of an intromittent organ [7,8] and show various extraordinary reproductive strategies including maternal dermatotrophy
and intrauterine feeding [9,10]. We investigated the potential for sperm storage in a species with high parental
investment (Ichthyophis cf. kohtaoensis, see Figure 1A). Apparently reproductive success is strongly dependent on environmental conditions
such as temperature and humidity. Under fluctuating conditions sperm storage in specialized
compartments in either the cloaca or the oviduct (or both) might be highly adaptive.
Yet, except for an historical observation of live sperm in the female oviduct more
than a century ago [11] nothing is known. We investigated this open question in field collected female Ichthyophis cf. kohtaoensis around the breeding season and found evidence of sperm presence and storage in caecilian
amphibians.

Findings

Paired oviducts are located lateral to the kidneys. Extending anteriorly to the heart
they terminate in small ostia, which receive the eggs and they join the cloaca posteriorly
(Figure 1B). Of seven female specimens collected prior to egg-laying four, contained mature,
yolky ovarian follicles (MD = 6.4 mm) and another three already had fertilized ova
located in the convoluted central part (pars convoluta, PC) of the oviduct (see Table 1). Stained slightly red to pink with Heidenhain’s Azan, tripartite spermatozoa with
a characteristic flattened head (about 11 μm), a mid-piece, and a single flagellum
were found within the posterior oviduct in six of seven vitellogenic females (see
Figure 1F–G). To elucidate this region we briefly describe the respective parts of the oviduct.
Following the relatively short (1/5) anterior part (pars recta), the substantially
convoluted pars convoluta (PC) constitutes more than half the oviduct and comprises
two gross morphological and histological different portions. At the level of the posterior
end of the ovaries, the posterior part (about ¼) of the PC is well demarcated in reproductively
active females by its diameter (about ~2,5 mm) and thick wall that is composed of
radially arranged, simple branched tubular glands made of voluminous secretory cells
(AB-, intensively PAS+) with well stained, basal nuclei and plasma containing neutral
mucopolysaccharids and granules. They are separated into several compartments by septs
of connective tissue and orifice into the oviductal lumen. Short prolonging mucosal
folds lined with columnar ciliated epithelium that forms crypts, further narrow the
lumen. Glands regress at about the level of the posterior end of the kidneys (Figure 1D, E) The remaining straight posterior-most portion resembles the uterine part (PU)
of the oviduct, characterised by a thick layer of smooth circular musculature and
radially projecting, longitudinal mucosal folds. Those are lined with multi-rowed
columnar ciliated epithelium, which is interspersed with non-ciliated secretory cells
(PAS+) and frequently divided by cleft like crypts regressing further posteriorly.
Oviductal sperm were restricted to a region between the middle of this posterior aglandular
PU up to the transition into the posterior part of the glandular convoluted PC (Figure 1C). Spermatozoa were neither found further anteriorly nor in the cloaca and were absent
in females collected after oviposition (Table 1).

Table 1.List of femaleIchthyophiscf.kohtaoensisexamined and details of their reproductive status

The amount and localization of sperm varies greatly between gravid and pre-ovulatory
females. In the latter, large and medium amounts of free spermatozoa were present
in the oviductal lumen of the anterior PU, but were found most abundantly in between
the mucosal folds and their epithelial crypts. Spermatozoa were aggregated in bundles
and similarly orientated with heads pointing to the ciliated epithelium (Figures 1D–F). Further anteriorly, additional sperm were found within the distal parts of the
simple branched tubular glands at the transition into, and within the posterior PC.
Again, spermatozoa were similarly orientated (Figure 1G), more or less closely packed and associated with transparent fluid (PAS+) indicating
secretions of the glands.

Three gravid females contained sperm restricted to the aglandular PU, however in decreased
numbers, compared to the pre-ovulatory condition (Figure 1H). Spermatozoa were rarely detected in the oviductal lumen. Sperm were located between
the most distal ends of the longitudinal folds and respective epithelial crypts, sometimes
enclosed by the epithelium and often occurring more individually.

Discussion

Given that oviductal sperm were detected in all females that were collected prior
to oviposition, we can refute cloacal sperm storage in Ichthyophis cf. kohtaoensis, which is in accordance with the report that no cloacal tubules are available for
sperm storage [12]. We present evidence that cloacal storage in dedicated spermathecae widespread among
the Salamandroidea [3,5], might be lacking in caecilians.

Following the anecdotal observation of living sperm in the posterior oviducts of a
single female caecilian, I. glutinosus[13], we are able to fully verify oviductal sperm in another oviparous species in this
genus. The presence of spermatozoa (1) in a distinct region (2) prior to and after
ovulation, and (3) with a consistent orientation is the first evidence of sperm storage
in caecilians. Sperm were found in two subsequent but morphologically different sites
in the posterior oviduct. The distal glandular tubules of the posterior PC could possibly
resemble distinct sperm storage tubules (SSTs) similar to seminal receptacles in the
oviducts of birds or squamates. In snakes and lizards, SSTs are compound tubular or
alveolar glands located anteriorly between the uterus and infundibulum [14-16]. Other squamates possess additional, vaginal SSTs and iguanid lizards such as the
green anoles, fully replaced anterior receptacles [17,18]. But the prominent glands of the posterior convoluted oviduct seen in female caecilians
differ from SSTs. Evidently sperm were particularly frequent in the elongated uterine
part between the mucosal folds and epithelial crypts. Oviductal sperm storage in the
absence of glands, but within deep, narrow furrows and “crypts” of longitudinally
folds is also known for several squamates such as red-sided garter snakes, Thamnophis sirtalis parietalis[19] and ground skinks, Scincella lateralis[20].

However, the general morphology of the posterior oviduct and anatomical site of sperm
presence in Ichthyophis cf. kohtaoensis more closely resembles the situation found in Ascaphus truei, so far the only other amphibian known to have oviductal sperm storage. In A. truei sperm is stored in simple tubular glands equipped with ciliated and secretory cells
of the elongated ovisac, the posterior-most part of the oviduct [4]. No generalized information about the exact physiological mechanisms of SSTs is available
so far [2]. Such receptacles of both squamates and Ascaphus truei are basically continuations
of the epithelium and especially in the latter they were identified as sites for sperm
residence rather than physiologically dedicated for storage [5,20]. Such a functional significance of the PU and tubules of the posterior PC serving
as temporal sperm receptacles after copulation, we would suggest for caecilians as
well. Because sperm were not found more anterior in the PC and are totally absent
after ovulation, an additional physiological border eventually represented by gland
secretions might exist preventing the passage of spermatozoa along the entire oviduct
just until ovulation. But the mechanisms of sperm release for fertilisation remain
unknown.

Ichthyophis cf. kohtaoensis likely reproduce annually or biannually [21]. Reproduction is highly correlated with the rainy season: in late May, shortly after
the onset of the monsoon, males and females were present on the breeding site. Thus,
females carrying large quantities of oviductal sperm likely would have copulated recently,
but because ovulation was not yet initiated, spermatozoa must remain viable to enable
fertilization. Because gravid females carrying fertilized eggs and fewer sperm have
been collected c. three weeks later we assume sperm storage for at least a few weeks,
which also confirms previous field observations of a copulation and subsequent oviposition
[22].

Functionally, sperm storage in caecilians would be advantageous if an additional clutch
could be fertilized from a single mating. However, evidently this scenario of long-term
storage is not applicable because oviductal sperm are absent after oviposition due
to the lack of distinct SSTs apart from the main tube, which indicates storage for
a single breeding season only. We favour the hypothesis that by further uncoupling
reproductive events females might optimize the timing of each event, because one advantage
of sperm storage is to reproduce in response to environmental variation [2].

Short-term storage in Ichthyophis cf. kohtaoensis might also be evolved in correlation with mating. Because subterranean caecilians
have a highly specialised olfactory organ, the tentacle, surface associated chemical
signalling might be involved in mate finding but currently no experimental evidence
exists for this hypothesis. Although reproductive cycles seem to be synchronous, males
and females encounter rates might be sufficiently low to favour the evolution of sperm
storage. Female investment in offspring is high including a large yolk supply (see
Figure 1A and B), the building of subterranean nests for egg-laying and long-term guarding
of the clutch (see also Figure 1A). Post-copulatory sperm storage facilitates maternal control in order to increase
reproductive success, fitness [23] and to assure previous investment in eggs. Possible multiple matings and relations
of post-copulatory female choice but also sperm competition with respect to the varieties
among the male copulatory organ remains to be studied.

Methods

We examined eleven adult females of Ichthyophis cf. kohtaoensis collected in north-eastern Thailand (Isan region, Mekong Valley, Khemmarat District,
Ubon Ratchathani Province, see [21]) during May-June and January-February (see details in Table 1) and fixed in an aqueous solution of 4% formaldehyde. Specimens were opened through
a medioventral incision and the reproductive status of the gonads was assessed under
a stereomicroscope. To detect sperm and to analyse the oviductal morphology, conventional
histology was applied to serial sections of oviducts of vitellogenic specimens and
the most cranial, caudal and middle oviduct of gravid females carrying oviductal eggs.
In addition the most posterior part including the cloaca of three females was studied
(see [12]). After dehydration samples were embedded in paraffin, serially sectioned (8–12 μm)
and stained following standard protocols for Heidenhain’s Azan, Alcian Blue (pH 2,5)
and classic PAS reaction using nuclear fast red for counterstaining [24]. We adopt the tripartite oviductal terminology of [25].

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

AK collected samples during field-work. AK and SK designed the research. SK carried
out histology and analysis in the laboratory. SK and AK interpreted the results and
wrote the manuscript. Both authors read, edited and approved the final version of
the manuscript.

Acknowledgements

Werner Himstedt kindly provided a photo in Figure 1. We thank Ingrid Weiß and Benjamin Weiss for assistance and support during histology.
Lennart Olsson, Hendrik Müller, Ingo Schlupp and Elke Valk gave constructive comments
on earlier versions of the manuscript. Marvalee Wake and an anonymous reviewer are
acknowledged for their constructive criticism and helpful suggestions, which perfected
the final manuscript. SK is supported by the Volkswagen Stiftung (grant initiative
“Evolutionary Biology”: 84 205). The Deutsche

Forschungsgemeinschaft (DFG grant Hi 306/5–1) provided financial support for the fieldwork
of AK. We would like to dedicate our contribution to Werner Himstedt in honour of
his long-term effort in studying the biology of caecilian amphibians.